The release of neurotransmitter at synapses is controlled by the number of synaptic vesicles that are ready for fusion with the plasma membrane in response to calcium influx. The aim of this proposal is to determine how the number of such readily-releasable vesicles is regulated and what effect this and other pools of vesicles can have on release. At synapses throughout the nervous system, ultrastructural information shows distinct pools of vesicles. A subset of vesicles is docked to the plasma membrane at the active zone, primed for fusion. Following exocytosis, this readily-releasable pool of vesicles must be replenished from a distinct reserve pool of vesicles clustered in the vicinity of the active zone. Slow replenishment will cause synaptic depression, while over-replenishment will lead to augmentation of transmitter release. Changing the size and rate of replenishment of vesicle pools thus offers a powerful mechanism for modulation of synaptic strength. While we understand in considerable detail what governs the fusion of docked vesicles, much less is known about what controls the dynamics of these distinct vesicle pools. Acutely isolated sensory hair cells are an excellent system for these studies since their vesicle pools are well characterized anatomically. Also, release occurs at the basal end of the cell body, offering easy access to the sites of transmitter release and exocytosis can be monitored directly by measuring the increase in cell membrane capacitance that occurs when vesicles fuse. Specific Aim 1 establishes the basic release properties of hair cells and tests the hypothesis that transmitter release is most efficient at a certain stimulation frequency, i.e., that transmitter release is tuned. Specific Aim 2 tests the hypothesis that the size of pools and the interaction between them control exocytosis. It tests whether calcium and/or protein kinases regulate traffic between vesicle pools. Specific Aim 3 tests the hypothesis that glutamate enhances its own release by modulating pool dynamics via presynaptic metabotropic receptors. The findings will significantly advance our understanding of synaptic transmission. By revealing novel mechanisms for the modulation of neurotransmitter release they may identify targets for therapeutic intervention in neurological diseases.